Project Summary Our Multi-PI research team recently reported that the delta isoform of casein kinase-1 (CK1?) is amplified and/or overexpressed in over a third of all breast cancers, and that CK1? activation is especially manifest in refractory forms of breast cancer such as triple-negative breast cancer (TNBC) that lacks targeted therapies. Further, we established that silencing CK1?, or inhibition of CK1? kinase activity with our nanomolar potent, highly selective small molecule dual inhibitor of CK1? and CK1?, specifically compromises the growth, survival and invasion of breast cancer cells that overexpress CK1?. Notably, CK1? inhibition also provokes tumor regression of TNBC, including lung metastatic TNBC and basal-like PDX breast cancer models, and without any overt side effects. Finally, we demonstrated that CK1? inhibition disables WNT/?-catenin signaling, a frequently activated yet heretofore undruggable pathway that is activated in a broad cast of human malignancies. Importantly, our new studies suggest roles for CK1? in TNBC metastasis, where we have now shown that CK1? signaling is necessary to sustain the expression of: (i) transcription factors that direct the epithelial to mesenchymal transition (EMT), including Zeb1 and Snail2; (ii) matrix metalloproteinases-1 (MMP1), MMP2 and MMP9 that control TNBC cell invasion; and (iii) the breast cancer stem cell (BCSC) factors Bmi1 and Sox9 that control self renewal. Finally, we have shown that our CK1? inhibitors have synergy with select in-clinic DNA-damaging chemotherapies used to treat TNBC. Collectively, these data support the hypotheses that CK1? is a driver of breast cancer metastases and that targeting CK1? will block and improve treatment of metastatic triple negative breast cancer. Accordingly, in Aim1 we will use an iterative and rigorous research operating plan (ROP) to improve the pharmacokinetic (PK) properties of our dual CK1?/CK1? inhibitors, and will develop and test the efficacy of CK1?-selective inhibitors, to deliver safety assessment candidates suitable for subsequent IND-enabling studies. This ROP includes tests of synergy of our inhibitors with in-clinic agents used to treat TNBC. Further, using genetic approaches and our CK1? inhibitors, in Aim 2 we will test roles for CK1? in each step of the metastatic cascade, including intravasation, extravasation, latency and/or establishment at the secondary site. Finally, in Aim 3, using phosphoproteomics, activity-based proteomic profiling, and RNA-seq we will identify and test the roles of downstream effectors of CK1? in controlling TNBC metastasis, and the EMT, MMPs and BCSC targets of CK1? signaling. These studies, and those in TNBC that we have generated that are resistant to our CK1?/? inhibitors, will define acute and chronic changes in components of the kinome that could be exploited for combination studies, to improve therapeutic response and block the emergence of resistance. We submit our research program will establish CK1? as a vulnerability to target metastatic triple negative breast cancer, and that the small molecule CK1?/? or CK1? inhibitors that we develop will have a major impact in the breast oncology clinic. !